Process for producing d-mannitol

ABSTRACT

High concentration of free cells of heterofermentative lactic acid bacteria (LAB) in a resting or slowly growing state are used to convert fructose into mannitol. Efficient volumetric mannitol productivities and mannitol yields from fructose are achieved in a process applying cell-recycle, continuous stirred tank reactor and/or circulation techniques with native LAB cells or with LAB cells with inactivated fructokinase gene(s). Mannitol is recovered in high yield and purity with the aid of evaporation and cooling crystallization.

FIELD OF THE INVENTION

[0001] This invention relates to the use of microorganisms, namelylactic acid bacteria (LAB), and concerns particularly a new process forthe bioconversion of fructose into mannitol with free, native orfructokinase inactivated cells in a resting or a slowly growing state.The invention also relates to the re-use of the cell biomass forsuccessive bioconversions.

BACKGROUND OF THE INVENTION

[0002] D-mannitol is a six-carbon sugar alcohol, which is about half assweet as sucrose. It is found in small quantities in most fruits andvegetables (Ikawa et al., 1972; Bär, 1985). Mannitol is widely used invarious industrial applications. The largest application of mannitol isas a food additive (E421), where it is used e.g. as a sweet tastingbodying and texturing agent (Soetaert et al., 1999). Crystallinemannitol is non-sticky, i.e. it prevents moisture absorption, and istherefore useful as coating material of e.g. chewing gums andpharmaceuticals. In medicine, mannitol is used as osmotic diuretic forintoxication therapy and in surgery, parenteral mannitol solutions areapplied to prevent kidney failure (Soetaert et al., 1999). Mannitol isalso used in brain surgery to reduce cerebral edema.

[0003] At present, commercial production of mannitol is done bycatalytic hydrogenation of invert sugar with the co-production ofanother sugar alcohol, sorbitol. Typically, the hydrogenation of a50/50-fructose/glucose mixture results in a 30/70 mixture of mannitoland sorbitol (Soetaert et al., 1999). Besides the fact that mannitol isthe by-product of the chemical production process and thus liable tosupply problems, it is also relatively difficult to separate fromsorbitol. In contrast to most sugars and other sugar alcohols mannitoldissolves poorly in water (13% (w/w) at 14° C. (Perry et al., 1997)).Cooling crystallization is therefore commonly used as a separationmethod for mannitol. However, according to Takemura et al. (1978) theyield of crystalline mannitol in the chemical process is still onlyapproximately 17% (w/w) based on the initial sugar substrates.

[0004] In order to improve the total yield of mannitol it would beadvantageous to develop a process with mannitol as the main product andwith no sorbitol formation. Some alternative processes based on the useof microbes have been suggested in the literature. Yeast, fungi, and LABespecially, are able to effectively produce mannitol withoutco-formation of sorbitol (Itoh et al. 1992). Among LAB onlyheterofermentative species are known to convert fructose into mannitol(Pilone et al. 1991; Axelsson, 1993; Soetaert et al. 1999). Speciesbelonging to the genera Leuconostoc, Oenococcus and Lactobacillusparticularly, have been reported to produce mannitol effectively. Inaddition to mannitol these microbes co-produce lactic and acetic acid,carbon dioxide and ethanol. These by-products are, however, easilyseparable from mannitol.

[0005] Soetaert and co-workers have studied the bioconversion offructose into mannitol with free cells of Leuconostocpseudomesenteroides ATCC-12291 (Soetaert et al., 1994). Using afed-batch cultivation protocol they reached a maximum volumetricproductivity of 11 g mannitol/L/h and a conversion efficiency ofapproximately 94 mole-%. Recently, Korakli et al. (2000) reported a 100%conversion efficiency with Lactobacillus sanfranciscensis LTH-2590.Other heterofermentative LAB reported to be good producers of mannitolinclude Leuconostoc mesenteroides, Oenococcus oeni, Lactobacillusbrevis, Lactobacillus buchneri and Lactobacillus fermentum (Pimentel etal., 1994; Salou et al. 1994; Erten, 1998; Soetaert et al. 1999).

[0006] In JP62239995, Hideyuki et al. (1987) used free cells of Lb.brevis. The volumetric mannitol productivity achieved in batchfermentation was 2.4 g/L/h.

[0007] EP0486024 and EP0683152 describe a strain named Lb. sp. B001 withvolumetric mannitol productivities of 6.4 g/L/h in a free cell batchfermentation (Itoh et al, 1992; Itoh et al., 1995).

[0008] More recently, Ojamo et al. (2000) have submitted a patentapplication for a process for the production of mannitol by immobilizedLAB. In this process the average volumetric mannitol productivity andconversion efficiency achieved were approximately 20 g/L/h and 85%,respectively. A low-nutrient medium was used which considerably lowersthe production costs. Immobilization also enables the re-use of cellbiomass for successive batch fermentations.

[0009] These inventions have not yet replaced the conventionalhydrogenation process. The free cell bioconversion processes describedto date are not entirely suitable for industrial scale production.Volumetric productivities in the range of 20 g/L/h, as achieved with theimmobilization process, should however, be adequate for profitableproduction. In order to further develop the features of thebioconversion alternative, factors such as equipment investment costs,robustness of the process, medium composition (raw material costs), andmannitol yields must be considered and improved. The goal of the presentinvention is to overcome the prior disadvantages, such as the lowproductivities obtained with the free cell bioconversion systems and thelow mannitol yields characteristic for all available bioconversionsystems. Thus, the goal of the present invention is to develop abioconversion process, which is feasible both technically andeconomically.

SUMMARY OF THE INVENTION

[0010] The present invention is accomplished to overcome thedisadvantages mentioned above. The present invention provides a processin which a high concentration of free cells of lactic acid bacteria isapplied to the bioconversion of fructose into mannitol. During thebioconversion phase the cells are kept in a resting or a slowly growingstate by supplementing to the fructose containing solution only minimalamounts of complex nutrients required for growth. The present inventiondescribes the use of an efficient, high-yield mannitol-producing strainin the process. The strain in question was identified by comparing themannitol production capabilities of different LAB species kept in aresting or slowly growing state. The present invention also provides anefficient, robust production process with productivities over 20 gmannitol/L/h. In addition, the process concept described here is simpleto apply in industrial scale, and because of the low-nutrient mediumused in it, the raw material costs are minimized. Furthermore, byinactivating the fructokinase gene a 100% yield of mannitol fromfructose is obtained.

[0011] The invention thus concerns a process for the production ofmannitol by bioconversion, which process comprises the steps of bringinga high initial concentration of free, mannitol-producing lactic acidbacterial cells into contact with a low-nutrient medium supplementedwith a substrate convertible into mannitol, and a cosubstrate, in abioreactor system; performing the bioconversion under conditionssuitable for converting said substrate into mannitol; separating thebacterial cells from the medium by filtration to obtain a cell-freesolution; recovering from the cell-free solution the mannitol produced;and reusing the separated bacterial cells in the bioreactor system.

[0012] Consequently, an object of the present invention is to provide asemi-continuous or a continuous process for the production of mannitol.One process alternative to accomplish this is the re-use of free cellbiomass in successive batch bioconversions as shown in FIG. 1. When theinitial fructose is depleted the cells are concentrated e.g. bytangential flow filtration (TFF), whereby the mannitol is removed fromthe bioconversion reactor in the cell-free permeate. The cellconcentrate is then diluted with fresh fructose-rich solution and a newbatch is started. During the bioconversion the cells are kept in aresting or slowly growing state.

[0013] Another embodiment of the present invention provides a processwhere a fructose-rich solution in a mixing reactor is circulated througha bioconversion reactor containing free cells in a resting or slowlygrowing state. The cells are kept in the bioconversion reactor bycell-recycle techniques (e.g. TFF; see FIG. 2) and the cell-freepermeate is re-circulated back to the mixing reactor. The volume in thebioconversion and mixing reactors is kept approximately constant.

[0014] A third embodiment of the present invention is a continuousprocess where a fructose-rich solution is added to a bioconversionreactor containing free cells in a resting or slowly growing state. Thecells are kept in the bioconversion reactor by cell-recycle techniques(e.g. TFF) and the mannitol-rich, cell-free permeate is directed todownstream processing via a recovery tank (FIG. 3). The volume of thebioconversion reactor is kept constant by continuous stirred tankreactor (CSTR) techniques (e.g. by level controller, calibrated feed andharvest pumps, or balancing the bioconversion reactor).

[0015] Furthermore, the present invention relates to the use of LAB inthe process. Several species can be used in the process with varyingyields and productivities (see Table 1 in Example 9). For instance,Leuconostoc pseudomesenteroides has a high productivity, but a yieldless than 80%. This is due to a strong leakage of fructose substrate tothe phospho-ketolase pathway via fructokinase-catalyzed phosphorylation.On the other hand, Lactobacillus sanfranciscensis gives a 100% mannitolyield from fructose, but is low in productivity (less than 0.5 g/L/h).To have both a high productivity and to maximize the yield, thefructokinase gene(s) is/are inactivated in the present invention in ahigh productivity species like Leuconostoc mesenteroides, Leuconostocpseudomesenteroides or Lactobacillus fermentum.

[0016] Consequently, further objects of the invention are bacterialstrains of the genus Lactobacillus or Leuconostoc, in which thefructokinase enzyme(s) is/are inactivated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1. Batch process alternative. Phase 1: Bioconversion. Phase2: Product recovery and cell concentration with tangential flowfiltration (TFF). Phase 3: Addition of fresh fructose-rich solution tothe concentrated cell suspension.

[0018]FIG. 2. Circulation process alternative. The cells are kept withinthe system consisting of the bioconversion reactor unit, the retentateside of the filtration unit and the circulation loop.Fructose-containing solution is pumped from the mixing reactor at thesame flow rate as permeate is added to the mixing reactor.

[0019]FIG. 3. Continuous process alternative. Fresh fructose-richsolution is prepared in the mixing reactor, which is then transferredinto the feed tank. Solution is added to and removed from thebioconversion reactor system, consisting of the bioconversion reactorunit, the retentate side of the filtration unit and the circulationloop, at the same flow rates.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The primary embodiment of the present invention is a process inwhich mannitol is produced by bioconversion from fructose with the aidof free native or fructokinase inactivated LAB cells kept in a restingor a slowly growing state. The volumetric mannitol productivities andmannitol yields from fructose for such a system are preferably above 10g/L/h and 90 mole-%, respectively.

[0021] The preferred substrate for the bioconversion is fructose.Sucrose can be used as well. In addition, glucose is preferred as aco-substrate for the production of NAD(P)H, which is needed as acofactor in the bioconversion of fructose into mannitol. Based on a 100mole-% bioconversion yield of fructose into mannitol, the preferredmolar ratio of fructose and glucose is 2:1. Typically amongheterofermentative lactic acid bacteria a varying fraction of fructosethat has been transported into the cells is phosphorylated byfructokinase-catalysis to form fructose-6-P and thus, channeled into thephosphoketolase pathway. The “leaking” fructose carbon skeleton is thenconverted stepwise into end products such as acetic and lactic acid,ethanol and carbon dioxide. When fructose is leaking to thephosphoketolase pathway and when the mannitol yield from fructose isless than 100 mole-%, it is preferable to increase the fructose toglucose ratio to avoid residual glucose concentrations. Preferredinitial concentrations of fructose and glucose vary from 50 to 200 g/Land 20 to 100 g/L, respectively. The upper limit of initial fructoseconcentration is usually set by the maximum solubility of mannitol atthe bioconversion conditions in question. An end concentration ofmannitol over the maximum solubility would result in crystallinemannitol to form in the bioconversion reactor, which preferably shouldbe avoided.

[0022] Instead of using high-purity fructose and/or glucose as thesubstrates, also respective compounds with a lower purity can be used asthe substrate for the cells. This is preferred in order to lower the rawmaterial costs, which are strongly influenced by the price of fructoseand glucose. Besides the sugars noted earlier, the bioconversion mediumalso needs to be supplemented at least with complex nitrogen sources,magnesium and manganese ions. The preferred complex nitrogen sources areyeast extract, preferably in initial concentrations of 0.1 to 1 g/L, andtryptone, preferably in initial concentrations of 0.2 to 2 g/L. Theconcentrations of magnesium and manganese ions are preferably in therange from 0.1 to 0.5 g/L and 0.01 to 0.1 g/L, respectively.Concentrations providing optimum mannitol production depend on thestrain in question and can therefore, deviate from the numbers shownabove. The magnesium and manganese ions can preferably be added in theform of respective sulphates. Alternative and less expensive complexnitrogen sources are e.g. soybean and cottonseed meal, corn steep liquor(CSL), yeast hydrolysates etc.

[0023] The preferred minimum concentration of free cells in thebioconversion reactor is 5 g dry cell weight/L. A value over 10 g/L ispreferred. The initial cell biomass production, which enables the firstbioconversion cycle to proceed, can be achieved by cultivating the cellsin a nutrient-rich growth medium, applying techniques such as batch,fed-batch, or CSTR cell-recycling. The cells are then concentrated tohigh cell densities, preferably 25 to 100 g dry cell weight/L, by e.g.tangential flow filtration (TFF) or centrifugation. Once the cells arein the bioconversion reactor, in the preferred concentrations mentionedabove, the same cells can be used for several successive batchbioconversions (see FIGS. 1 and 2). Hence, the processes according toalternatives shown in FIGS. 1 and 2 of the present invention aresemi-continuous.

[0024] The bioconversion and the mixing reactors are preferably agitatedvessels with the possibility to measure and control on-line thetemperature and pH of the bioconversion medium. Pressure indicatorsshould preferably also be available. The carbon dioxide formed duringthe bioconversion is preferably released via the headspace either in thebioconversion or in the mixing reactor or both. The vessels arepreferably made out of food-grade stainless steel material and thesystem should preferably be suitable for aseptic process protocols.Several reactors may be used in series and/or in parallel. For instance,nitrogen flushing of the media can be used to improve the mannitolyields from fructose and the CO₂ removal from the bioreactors.

[0025] The temperature and pH of the bioconversion medium shouldpreferably be controlled either in both the bioconversion and the mixingreactor or only in one of the reactors. The temperature can be adjustedeither with e.g. water or steam, whereas the pH can be adjusted withe.g. NaOH, KOH, NH₄OH, HCl or H₂SO₄ solutions. The temperature and pHshould preferably be adjusted within the respective optimum values inorder to provide maximum mannitol productivity.

[0026] A suitable microorganism, in its native form, should preferablyexpress mannitol dehydrogenase activity and produce mannitol as its mainmetabolite. Among suitable microorganisms are Leuconostoc mesenteroides,Leuconostoc pseudomesenteroides, Lactobacillus brevis, Lactobacillusbuchneri, Lactobacillus fermentum, Lactobacillus sanfranciscensis, andOenococcus oeni. The preferred species is Leuconostoc mesenteroides andespecially strain ATCC-9135. The present invention is, however, notlimited to these microorganisms. The present invention also refers tomicroorganisms with activities similar to those mentioned above. Alsomicroorganisms derived, by e.g. recombinant techniques, frommicroorganisms mentioned above or from microorganisms with activitiessimilar to those mentioned above, may be used in the process.

[0027] If the concentration of free cells in the bioconversion reactoris increasing too much so that e.g. the productivity is decreasing froma normal value, a suitable volume of the cell suspension can be removedfrom the system. In the batch version of the present invention (FIG. 1)this is preferably done before the fresh bio conversion medium is addedto the high cell density suspension, in order to start a newbatch-cycle. In the circulation version of the present invention (FIG.2) the removal is preferably done while the mixing vessel is emptiedafter fructose depletion and then refilled with fresh bioconversionmedium. In the continuous version of the present invention (FIG. 3) e.g.the dilution rate and the contents of the feeding solution are used tocontrol the production of mannitol. If it is necessary to remove cellsfrom the continuous bioconversion reactor, it can be done applying e.g.TFF techniques.

[0028] While a microfiltration membrane or a large ultrafiltrationmembrane (e.g. 1000 kDa) is used in the TFF equipment for cellseparation, it is not expected that any other component would beconcentrated to harmful levels in the system, while these are mostlikely removed from the bioconversion reactor with the permeate oralternatively consumed by the cells.

[0029] The inactivation of the fructokinase activity is accomplishedeither by classical mutagenesis or by targeted gene inactivationtechniques. Classical mutagenesis is done by treating growing cells ofLAB with 1-methyl-3-nitro-1-nitrosoguanidine and selecting for bacteria,which cannot grow on fructose as the sole carbon source. The obtainedmutants are further tested for their ability to import fructose into thecell to assure that the growth defect on fructose is not caused by amutation present in fructose permease. The fructose transport isverified using radioactively labeled fructose in the growth medium anddetecting the radioactivity in separated washed cells. Alternatively,the transport of fructose can be indirectly confirmed by measuring theconversion of fructose to mannitol in growth medium containing fructose.

[0030] The targeted inactivation of the fructokinase gene is done eitherby disrupting or by deleting the fructokinase gene. The inactivationplasmids for both purposes are constructed using a vector plasmid withtemperature sensitive replication origin to enhance the integrationevent to the bacterial chromosome. One example of this kind of plasmidis pGhost4, which is a wide host-range plasmid, capable of replicatingin many Gram-positive bacteria (Biswas et al., 1993). In the first phasethe inactivation plasmid is transferred to LAB by electroporation andtransformants are selected at a permissive temperature using antibioticselection. In the second phase, integration of the plasmid to thebacterial chromosome is achieved by growing the transformants at anon-permissive temperature to plasmid replication, using stillantibiotic selection.

[0031] In the disruption construct an internal fragment of thefructokinase gene is cloned to the vector plasmid and integration at thefructokinase locus will interrupt the coding sequence and thus preventthe formation of an active fructokinase enzyme. In the case of targeteddeletion of the fructokinase gene, integration of the deletion plasmidin the second phase does not disrupt the coding sequence, but createstwo regions of homologous sequences, which serve as excision sites inlater steps. These regions determining the excision sites are cloned inthe deletion plasmid in a consecutive order and all DNA sequencesbetween these regions will be deleted when homologous recombinationoccurs. Also all plasmid sequences, together with the antibioticresistance gene, will be removed from the bacterial chromosome. Afterintegration of the deletion plasmid the transformant bacteria are grownwithout antibiotic and clones sensitive to antibiotic are selected andtested for growth on fructose. The clones that cannot grow on fructoseas sole carbon source are selected. The conversion of fructose tomannitol will be determined, and also the growth on the same substrates,used for native LAB strains, will be tested.

[0032] Mannitol is the main bioconversion product of the presentinvention. Other bioconversion products, which are dissolved in themedium, are e.g. acetic and lactic acid, and ethanol. Most of the carbondioxide in the liquid medium is preferably removed from the system asgaseous carbon dioxide through agitation and/or nitrogen flushing of themedium. The liquid product solution is separated from the cells by TFF,as shown in FIG. 1 (no additional cell separation step is needed in theother two process alternatives of the present invention). The rest ofthe product recovery process comprises of the following unit operations:concentration, crystallization, separation, drying, and homogenization.Alternatively also other metabolites formed, besides mannitol, can berecovered from the bioconversion medium.

[0033] The concentration of the liquid product solution is preferablydone by evaporation. The heated concentrate is then transferred to acooling crystallization unit, where mannitol crystals fall out when thetemperature of the solution is decreased. Next the crystals areseparated from the mother liquor by a drum separator and the crystalsthereby collected (crystals A). The mother liquor is either added to thenext recovery cycle or re-crystallized separately (crystals B).Alternatively, the mother liquid, if containing residual fructose, canbe recycled back to the bioconversion step. The crude crystals (A and B)are dissolved in hot water, where after the solution is re-crystallizedin a cooling crystallization unit. After a second drum separation stepthe white crystals are dried in a vacuum or under-pressure oven.Finally, if needed, the dry crystals are homogenized by a suitablemethod. According to the protocol presented above the total mannitolrecovery yield and crystal purity achieved, is preferably 50 to 100mass-%, and 95 to 100 mass-%, respectively.

EXAMPLE 1

[0034] Production of cells for the bioconversion phases 1

[0035] A bench-top bioreactor containing 9.7 L of nutrient-richfermentation medium (Soetaert et al., 1999) was inoculated with 300 mLof a 16-h cell culture of Leuconostoc pseudomesenteroides ATCC-12291grown in an inoculation medium (Soetaert et al., 1999). The temperatureof the growth medium (10 L) was set first at 20° C. and after 56 hraised to 25° C. The pH was controlled at 5.0. The solution was slowlyagitated.

[0036] After about 66 hours the cultivation was stopped and the cellsrecovered by tangential flow filtration (Pellicon® 2 Mini Holder andBiomax® 1000 (V screen) membrane, Millipore Corp., USA). From an initialvolume of 10.9 L (˜3 g dry cell weight/L) a 0.7-L cell concentrate (˜47g dry cell weight/L) was obtained by this filtration technique. Thecell-free permeate (10.2 L) could thereafter be used for study ofmannitol recovery. The cell concentrate can be used as the initialbiomass for the processes described in Examples 3-5.

[0037] The volumetric mannitol productivity of this free cell processwas 1.7 g/L/h.

EXAMPLE 2

[0038] Production of cells for the bioconversion phases 2

[0039] A bench-top bioreactor containing 1.9 L of MRS growth medium (40g/L glucose) was inoculated with 100 mL of a 10-h cell culture ofLeuconostoc mesenteroides ATCC-9135 also grown in a MRS growth medium(30 g/L glucose). The temperature and pH of the growth medium (2 L) wereset at 30° C. and 6.0, respectively. The solution was slowly agitated.

[0040] About 9.5 hours later the cells were harvested by centrifugation.The cell pellet was then suspended in a fresh bioconversion medium (SeeExamples 3-5).

EXAMPLE 3 Production of mannitol by bioconversion in a batch mode (FIG.1)

[0041] The cell pellets obtained in Example 2 was suspended with freshbioconversion medium and transferred aseptically into a bioconversionreactor. The total volume of the solution was 425 mL and it had thefollowing initial composition: 100 g/L fructose, 50 g/L glucose, 1 g/Ltryptone, 0.5 g/L yeast extract, 2.62 g/L K₂HPO₄.3H₂O, 0.2 g/L MgSO₄,and 0.01 g/L MnSO₄. The cell concentration during the bioconversion wasapproximately 10 g dry cell weight/L.

[0042] The temperature control was set at 30° C. and the pH wascontrolled at 5.0 with 3 M NaOH. The solution was slowly agitated.

[0043] After 4.5 hours of bioconversion time the cells had consumed allof the sugars and the experiment was ended. The average volumetricmannitol productivity for the process was 20.7 g/L/h. The mannitol yieldfrom fructose was 91.2 mole-%.

[0044] Furthermore, the product solution and cells can be separated bye.g. TFF, and the cell concentrate re-used in successive batchbioconversions according to the process description in FIG. 1.

EXAMPLE 4

[0045] Production of mannitol by bioconversion with circulation (FIG. 2)

[0046] The experiment set up is shown in FIG. 2. The cell pellets,obtained as described in Example 2, were suspended in freshbioconversion medium lacking the sugars and transferred aseptically tothe bioconversion reactor unit. The volume in the bioconversion reactorunit was 0.4 L. A TFF unit (Pellicon® 2 Mini Holder and Biomax® 1000 (Vscreen) membrane, Millipore Corp., USA) was attached to thebioconversion bioreactor unit and the permeate flow was lead to a mixingreactor. The mixing reactor (volume 1.0 L) was standing on a balance andthe mass of the reactor was kept constant by circulating medium back tothe bioconversion reactor unit. The total volume of the whole system was1.5 L and the medium had the following initial composition: 100 g/Lfructose, 50 g/L glucose, 1 g/L tryptone, 0.5 g/L yeast extract, 2.62g/L K₂HPO₄.3H₂O, 0.2 g/L MgSO₄, and 0.01 g/L MnSO₄. The cellconcentration in the bioconversion reactor was 8.7 g dry cell weight/L.

[0047] The temperature and the pH were controlled both in thebioconversion reactor and in the mixing reactor units. The temperaturecontrol was set at 30° C. and the pH was controlled at 5.0 with 3 MNaOH. Mixing was applied in both reactors.

[0048] After 9 hours of bioconversion time the cells had consumed all ofthe sugars and the experiment was ended. The average volumetric mannitolproductivity for the process was 21.6 g/L/h. The mannitol yield fromfructose was 94.0 mole-%.

EXAMPLE 5

[0049] Production of mannitol by bioconversion in a continuous reactor(FIG. 3)

[0050] The experiment set up is shown in FIG. 3. The cell pellets,obtained as described in Example 2, were suspended in freshbioconversion medium lacking the sugars and transferred aseptically tothe bioconversion reactor unit. A TFF unit (Pellicon® 2 Mini Holder andBiomax® 1000 (V screen) membrane, Millipore Corp., USA) was attached tothe bioconversion bioreactor unit and the permeate flow was lead to arecovery tank. The total volume in the bioconversion reactor unit,retentate side of the filtration unit, and in the circulation loop was1.0 L. The bioconversion reactor unit was standing on a balance and themass of the reactor was kept constant by adding fresh medium from a feedtank. The feeding solution following initial composition: 25 g/Lfructose, 12.5 g/L glucose, 1 g/L tryptone, 0.5 g/L yeast extract, 2.62g/L K₂PO₄.3H₂O, 0.2 g/L MgSO₄, and 0.01 g/L MnSO₄. The cellconcentration in the bioconversion reactor, at dilution rate 0.68 1/h,was approximately 6.9 g dry cell weight/L.

[0051] The temperature control was set at 30° C. and the pH wascontrolled at 5.0 with 3 M NaOH. The reactor was slowly agitated. Avolumetric mannitol productivity of 12.5 g/L/h was achieved. Themannitol yield from fructose was 93.0 mole-%.

EXAMPLE 6

[0052] Inactivation of the gene encoding fructokinase by randommutagenesis

[0053] Chemical mutagenesis of L. pseudomesenteroides ATCC-12291 wasdone using log-phase cells (OD₆₀₀ 1.0) grown in M17 supplemented with 1%glucose (GM17). Cells washed with 50 mM sodium phosphate buffer, pH 7,were treated with 1-methyl-3-nitro-1-nitrosoguanidine, 0.5 mg/ml, for40-50 min, at room temperature, and washed three times with the bufferabove. Washed cells were incubated in GM17, for 1 hour, at 30° C., andplated on GM17 agarose, incubated 2 days at 30° C. Colonies on GM17plates were replica-plated on a chemically defined medium (CDM; Anon.,2000) supplemented with either 1% glucose or 1% fructose. After 2 daysof incubation at 30° C. colonies growing on glucose, but not onfructose, were selected. Conversion of fructose to mannitol willindicate that the fructose permease is not affected by the mutagen. Thefructokinase inactivated production strain, which was able to convertfructose to mannitol, was named BPT-143. The strain was depositedaccording to the Budapest Treaty at the Deutsche Sammlung vonMikroorganismen und Zeilkulturen, GmbH, Mascheroder Weg 1b, D-34124Braunschweig, Germany on Nov. 13, 2001 with the accession number DSM14613.

EXAMPLE 7

[0054] Inactivation of the gene encoding fructokinase by directedmutagenesis

[0055] Inactivation plasmid for disrupting the fructokinase gene(s) ofLb. fermentum is constructed by joining an internal fragment of afructokinase gene between suitable restriction sites of pGhost4. Theligation mixture is electroporated to Lactococcus lactis, transformantsare incubated for 1 day, at permissive temperature, 30° C., usingerythromycin (Em, 5 μg/ml) and screened by PCR with pGhost4-specificprimers. Recombinant plasmids, containing the internal fragment offructokinase gene, are isolated and electroporated to Lb. fermentum.Transformants are incubated anaerobically, for 1 day, at 30° C., andverified by PCR with the previously mentioned primers. Clones carryingthe recombinant plasmids selected for the integration experiments aregrown over night, at 30° C., in MRS growth medium supplemented with 5μg/ml Em. These cell suspensions are used as inoculate for new culturesgrown for 5 hours at 42° C. in same medium. Then the cell suspensionsare diluted 1:100 000, plated on MRS-Em, and incubated for 2 days at 42°C. Colonies arising in the presence of Em at 42° C. will have adisruption plasmid integrated to the chromosome at the fructokinaselocus. Disruption of the fructokinase gene(s) will result in reducedfructokinase activity of the disruption transformants compared to thewild type Lb. fermentum grown in MRS or CDM supplemented with differentsugars (sucrose, fructose, lactulose, maltose, galactose or ribose) and5 μg/ml Em. Disruption of the fructokinase gene(s) is confirmed bySouthern blotting of the chromosomal DNA isolated from the clones withreduced fructokinase activity.

[0056] Fructokinase genes are deleted using the following protocol. Two0.5 kb fragments amplified by PCR from Lb. fermentum chromosome,surrounding the targeted deletion site, are ligated to pGhost4. Theligation mixture is electroporated to L. lactis, transformants areincubated for 1 day at permissive temperature, 30° C., usingerythromycin (Em, 5 μg/ml) and screened by PCR with pGhost4-specificprimers. Plasmids containing the cloned fragments are isolated andelectroporated to Lb. fermentum. Transformants are incubatedanaerobically on MRS-Em plates for 1 day at 30° C. and resultingcolonies are verified by pGhost4-specific primers to ensure the presenceof the recombinant plasmids and correct insert sizes. Raising thetemperature as described for the disruption plasmids will result inintegration of the recombinant plasmid to the chromosome. Sites of theintegration are confirmed by Southern blotting of chromosomal DNAisolated from the integrant strains. The Lb. fermentum carrying anintegrated recombinant plasmid at a fructokinase locus is then grownwithout Em, at 42° C., for 100 generations and plated on MRS without Em.Omission of the antibiotic will result in dissociation of the integratedplasmid from the chromosome. Depending on the recombination site eitherrestoration of the wild type or deletion of a fructokinase gene willhappen. In both cases all foreign DNA will be removed from thechromosome. Em-sensitive clones are detected after replica plating onMRS with and without Em. Among the Em-sensitive clones those withreduced fructokinase activity are selected. Deletion of the fructokinasegene is confirmed by Southern blotting the chromosomal DNA isolated fromthe deletion strains.

EXAMPLE 8

[0057] Production of mannitol by L. pseudomesenteroides with inactivatedfructokinase gene (random mutagenesis)

[0058]L. pseudomesenteroides ATCC-12291 and the clone DSM 14613 (BPT143) with inactivated fructokinase gene (see Example 6) were tested formannitol production in parallel experiments. The growth medium had thefollowing composition: 20 g/L fructose, 10 g/L glucose, 10 g/L tryptone,5 g/L yeast extract, 2.62 g/L K₂HPO₄.3H₂O, 0.4 g/L MgSO₄, and 0.02 g/LMnSO₄. The temperature and pH was set at 30° C. and 5.0, respectively.The bioconversion time was 8 hours. The mannitol yields from fructosefor the native strain and the clone were 73.7 mole-% and 85.7 mole-%,respectively. Also, a 25% improvement in volumetric mannitolproductivity was observed.

EXAMPLE 9

[0059] Comparison of mannitol production capacity of lactic acidbacteria in a resting or slow-growing state

[0060] Pre-cultures of three of the most promising strains (preliminarycomparison studies not shown) were grown in MRS growth medium. The cellsuspensions were centrifuged and the cell pellets washed in 0.2 Mphosphate buffer (pH 5.8). After an additional centrifugation separationthe cell pellets were resuspended in the same buffer. The concentratedcell suspensions (50 mL per strain) were added to bioreactors containing450 mL of a bioconversion medium. After addition the composition of thesolution was the following: 20 g/L fructose, 10 g/L glucose, 0.5 g/Ltryptone, 0.25 g/L yeast extract, 2.62 g/L K₂HPO₄.3H₂O, 0.2 g/L MgSO₄,and 0.01 g/L MnSO₄.

[0061] The temperature and pH of the bioconversion medium were set at30° C. and 5.0, respectively. The bioconversion media were slowlyagitated. The key results are shown in Table 1. TABLE 1 The volumetricmannitol productivities (r_(mtol)) and mannitol yields from glucose(Y_(mtol/fru)) after 8 hours of bioconversion time. r_(mtol)Y_(mtol/fru) Strain: (g/L/h) (mole/mole) Leuconostoc mesenteroidesATCC-9135 2.3 97.8 Leuconostoc pseudomesenteroides ATCC-12291 1.5 79.6Lactobacillus fermentum NRRL-1932 1.0 86.1

EXAMPLE 10

[0062] Recovery of mannitol

[0063] The cell-free permeate, described in Example 1, was concentratedto approximately 250 g mannitol/L by evaporating with a Rotavapor unit.The concentrate (T=35° C.) was transferred into a coolingcrystallization unit and the temperature was linearly (15 h) decreasedto 5° C. The solution was slowly agitated. The crystals were separatedby filtration and the mother liquor was re-crystallized as describedabove.

[0064] The wet crystals from the first and the second cycle werecombined and dissolved in distilled water (T=45° C.). The mannitolconcentration of the solution was approximately 300 g/L. The solutionwas transferred into a cooling crystallization unit and the temperaturewas linearly (15 h) decreased to 5° C. The crystals were separated byfiltration and finally, the wet crystals were dried overnight at 60° C.

[0065] The recovery yield was about 55 mass-% and the purity above 99.5mass-%. The mannitol found in the washing solution gained from the lastcrystallization step can be re-used as part of the washing solution inthe next recovery cycle. Adding this hypothetical amount of mannitol tothe crystals obtained in the first recovery cycle a final recovery yieldof about 71% was achieved.

[0066] Deposited microorganisms

[0067] The following microorganism was deposited according to theBudapest Treaty at the Deutsche Sammlung von Mikroorganismen undZellkulturen, GmbH, Mascheroder Weg 1b, D-34124 Braunschweig, Germany.Microorganism Accession number Deposit date Leuconostoc pseudo- DSM14613 Nov. 13, 2001 mesenteroides BPT-143

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1. A process for the production of mannitol by bioconversion, comprisingthe steps of bringing a high initial concentration of free,mannitol-producing lactic acid bacterial cells into contact with alow-nutrient medium supplemented with a substrate convertible intomannitol, and a cosubstrate, in a bioreactor system, performing thebioconversion under conditions suitable for converting said substrateinto mannitol, separating the bacterial cells from the medium byfiltration to obtain a cell-free solution, recovering from the cell-freesolution the mannitol produced, and reusing the separated bacterialcells in the bioreactor system.
 2. The process according to claim 1,wherein the bacterial cells are native lactic acid bacterial cells. 3.The process according to claim 2, wherein the bacterial cells are of thestrain Leuconostoc mesenteroides ATCC-9135.
 4. The process according toclaim 1, wherein the bacterial cells are fructokinase-inactivated lacticacid bacterial cells.
 5. The process according to claim 4, wherein thecells are of the strain Leuconostoc pseudomesenteroides BPT143 (DSM14613).
 6. The process according to claim 1, wherein the substrate isfructose.
 7. The process according to claim 1, wherein the cosubstrateis glucose.
 8. The process according to claim 1, wherein thebioconversion is performed until at least 70%, preferably 90% or more ofthe said substrate has been consumed.
 9. The process according to claim1, wherein an average volumetric mannitol productivity of at least 10g/L/h is achieved.
 10. The process according to claim 1, wherein thebioconversion is performed as a batch process in a bioreactor systemcomprising a bioconversion reactor unit and a filtration unit.
 11. Theprocess according to claim 10, comprising the steps of separating thebacterial cells from the medium after the bioconversion step, andreusing said cells in successive bioconversions.
 12. The processaccording to claim 1, wherein the bioconversion is performed as acirculation process in a bioreactor system comprising a bioconversionreactor unit, a filtration unit and a mixing reactor unit.
 13. Theprocess according to claim 12, wherein the bacterial cells arecirculated between the bioconversion reactor unit and the filtrationunit.
 14. The process according to claim 12 or 13, comprising the stepsof leading the cell-free solution obtained by the filtration into themixing reactor, and transferring the solution from the mixing reactorback to the bioconversion reactor unit.
 15. The process according to anyone of the claims 12 to 14, comprising emptying the mixing reactor afterthe bioconversion step, and re-filling it with said low-nutrient mediumsupplemented with said substrate and co-substrate, to run successivebio-conversions reusing said bacterial cells.
 16. The process accordingto claim 1, wherein the bioconversion is performed as a continuousprocess in a bioreactor system comprising a mixing tank, a feed tank, abioconversion reactor unit, a filtration unit and a recovery tank. 17.The process according to claim 16, wherein the bacterial cells arecirculated between the bioconversion reactor unit and the filtrationunit.
 18. The process according to claim 16 or 17, comprising the stepsof feeding the bioconversion reactor unit continuously with saidlow-nutrient medium supplemented with said substrate and co-substrate,and removing the cell-free solution gained by filtration from thebioreactor to withhold a constant volume in the bioconversion reactorunit.
 19. The process according to any one of the claims 10 to 18,wherein the filtration unit is a tangential flow filtration unit. 20.The process according to any one of the claims 10 to 18, wherein saidbioconversion is run in series or in parallel.
 21. A bacterial strain ofthe genus Lactobacillus or Leuconostoc, in which the fructokinaseenzyme(s) is/are inactivated.
 22. The bacterial strain according toclaim 21, in which the fructokinase enzyme(s) is/are inactivated byrandom mutagenesis.
 23. The bacterial strain according to claim 22,which is Leuconostoc pseudomesenteroides BPT143 (DSM 14613).
 24. Thebacterial strain according to claim 21, in which the fructokinaseenzyme(s) is/are inactivated by directed mutagenesis.
 25. Use of abacterial strain of the genus Lactobacillus or Leuconostoc, in which thefructokinase enzyme(s) is/are inactivated, for producing mannitol.